Uses of inorganic pyrophosphates

ABSTRACT

Pharmaceutical compositions comprising an inorganic pyrophosphate (PPi) in a form that renders the PPi accessible to ABC proteins when administered to a subject in vivo, as well as use of the compositions for treating subjects having a disease or disorder associated with inappropriate or inadequate ABC protein activity (e.g., cystic fibrosis, multi drug resistance, Zellweger&#39;s Syndrome) is disclosed.

BACKGROUND OF THE INVENTION

ATP-binding cassette (ABC) proteins are an ancient class of membranetransporters, found throughout phylogeny in eubacteria, archezoa andmetakaryota (Gros, P. et al. (1986) Cell 47:371-370; Chen, C. J. et al.(1986) Cell 47:381-389; Kuchler, K. et al. (1989) EMBO J. 8:3973-3984;Riordan, J. R. et al. (1989) Science 245:1066-1073; Hyde, S. et al.(1990) Nature 346:362-365). During evolution, ABC proteins have becomespecialized in uptake and secretion, intracellular transport, celldetoxification and signaling and translocate highly diverse compoundsacross cell membranes, such as ions, amphiphiles, sugars, peptides andproteins.

The diverse functions and substrate specificities are accomplished by acommon protein architecture (Hyde, S. et al. (1990) Nature 346:362-365).Each of the 1-4 functional units of an ABC protein consists of ahydrophobic domain of six membrane spanning segments and a hydrophiliccytoplasmic domain which is able to bind ATP. Three 20-45 amino acidsequence motifs in the nucleotide binding folds (NBF) are highlyconserved among ABC transporters.

The ABC protein family includes yeast (STE6 gene product ), bacterial(haemolysin transport protein; hisP, malK, oppD and pstB proteins whichare involved in ATP-dependent transport of specific molecules throughbacterial inner cell membrane) and mammalian proteins. Four mammalianfamily members are currently known (PMP70, MHC-linked transport protein,P-glycoprotein, and the cystic fibrosis transmembrane conductanceregulator protein (CFTR)).

PMP70

The peroxisomal membrane protein (PMP70) is one of the major integralmembrane proteins of rat liver peroxisomes (Kamijo, K. et al. (1990) J.Biol. Chem. 265(8):4534-4540). It is believed that PMP70 may be involvedin an active transport process through the peroxisomal membrane. One ofthe proposed functions for PMP70 protein is a transport of acyl-CoAcompounds across peroxisomal membrane. Peroxisomes are cellularorganelles bounded by a single membrane, and are observed in almost alltypes of eucaryotic cells. Although the physiological significance ofperoxisomes has remained elusive, diseases caused by a generaldysfunction of peroxisomes, including Zellweger syndrome, infantileRefsum disease, hyperpipecolic acidaemia, and neonataladrenoleukodystrophy, have recently been recognized (Schutgens, R. B. H.et al. (1986) Eur. J. Pediatr. 144:430-440). The severe clinicalmanifestation of these diseases indicate the indispensable roles ofmammalian peroxisomes.

In cells of patients with Zellweger syndrome, peroxisomes aremorphologically absent (Goldfischer, S. et al. (1973) Science182:62-64). Biogenesis of peroxisomes is apparently impaired in thisdisorder; the cause may be defects in the protein machinery of theperoxisomes (Santos, M. J. et al. (1988) Science 239:1536-1538). It hasbeen further postulated that the primary biochemical lesion is at thelevel of the biosynthesis of a protein essential for the import ofperoxisomal enzymes from the cytoplasm into the peroxisomes whichutilizes adenosine triphosphate during import (Schutgens et al. (1986)Eur. J. Pediatr. 144:430-440; Imanaka, T. (1987) J. Cell Biol.105:2915).

MHC-linked (TAP) Transport Proteins

The MHC-linked (TAP) transport proteins appear to be required forantigen processing and presentation (Deverson, E. V. (1990) Nature348:738-740; Monaco, J. J. et al. (1990) Science 250:1723-1726; Spies,T. et al., (1990) Nature 348:744-747). This MHC-linked transport proteinmay deliver intracellularly degraded antigens to the endoplasmicreticulum for binding to the class I major histocompatability molecules(Spies and DeMars (1991) Nature 351:323-324). Several mutant cell lineshave been described in the literature, human B lymphoblastoid cell line(LCL) mutant and mutant murine cell line RMA-S, which have lost theability to form peptide-MHC complexes. Since antigen that is derivedfrom the cytoplasm must cross a lipid bilayer in order to associate withthe external portion of MHC class I molecules, the most likely defect inthese cells may be an inability to translocate the antigen from thecytoplasm into the appropriate membrane bound compartment where thisassociation normally takes place. The region of the MHC implicated inthese mutations contains the genes for the MHC-linked transport protein.

P-glycoprotein

P-glycoprotein is primarily expressed at epithelial and endothelialsurfaces (Thiebaut, F. et al., (1987) PNAS USA 84:7735-7738) and isassumed to play an essential role in absorption and/or secretion.P-glycoprotein is believed to have two distinct and independentfunctions. P-glycoprotein is an active transporter which pumpshydrophobic drags out of cells, reducing their cytoplasmic concentrationand therefore toxicity. Thus, one function of P-glycoprotein is toeliminate toxic metabolites or xenobiotic compounds from the body(Endicott and Ling (1989) Annu. Rev. Biochem. 58:137-171; Croop, J. M.et al. (1988) J. Clin. Invest. 81:1303-1309; Gottesman and Pastan (1988)J. Biol. Chem. 263:12163-12166; Van der Bliek and Borst (1989) Adv.Cancer Res. 52:165-203).

P-glycoprotein has also been associated with a volume-regulated chloridechannel activity. It has been reported that expression of P-glycoproteingenerates volume-regulated, ATP-dependent, chloride-sensitive channels,with properties similar to channels characterized previously inepithelial cells (Valverde, M. A. (1992) Nature 355:830-8330).Therefore, P-glycoprotein is also believed to be involved in nutrientabsorption in intestinal villus or in the placental cells at the site ofmaternal-foetal exchange (Trezise, A. E. O. (1992) EMBO J.11(12):4291-4303). Unlike CFTR channels which are regulated by cyclic,P-glycoprotein is volume regulated, (e.g., swelling induced activation).

Overexpression of P-glycoprotein confers the phenotype of multidrugresistance (mdr; Gros, P. et al. (1986) Nature 323:728-731) which maycause failure of chemotherapy in cancer (Goldstein, L. J. et al. (1989)J. Natl. Cancer Inst. 81:116-124). The selection and proliferation ofdrug-resistant tumor cells represents a major cause of failure in thechemotherapeutic treatment of human tumors. Tumors initially sensitiveto a cytotoxic agent often recur and are resistant to a broad spectrumof chemotherapeutic drugs (Wittes and Golden (1986) Cancer Treat. Rep.70:105-125). From the study of highly drug-resistant cell lines derivedin vitro, it is generally agreed that a net decrease of theintracellular concentration of drug underlies the multidrug-resistantphenotype (Bhalla, K. et al. (1985) Cancer Res. 45:3657-3662). It is nowbelieved that P-glycoprotein binds the drugs to which a mdr cell isresistant or collaterally sensitive (Busche, R. et al. (1989) Mol.Pharmacol. 35:414-421; Busche, R. et al. (1989) Eur. J. Biochem.183:189-197) and hydrolyzes ATP (Hamada and Tsuruo (1988) J. Biol. Chem263:1454-1458).

Cystic Fibrosis Transmembrane Conductance Regulator protein (CFTR)

The cystic fibrosis transmembrane conductance Regulator protein (CFTR)is a 1480 amino acid protein containing two membrane-spanning domains(MSDs), two nucleotide binding domains (NBDs) and a unique R domain,that functions as a chloride channel regulated by phosphorylation and bynucleoside triphosphates.

Cystic Fibrosis (CF) is the most common fatal genetic disease in humans(Welsh M. J. et al. in The Metabolic Basis of inherited Diseases, Vol.III, pp. 3799-3876 (Striver, C. R. et al. eds., McGraw-Hill, New York(1995)). Approximately one in every 2,500 infants in the United Statesis born with the disease. At the present time, there are approximately30,000 CF patients in the United States. Despite current standardtherapy, the median age of survival is only 26 years. Disease of thepulmonary airways is the major cause of morbidity and is responsible for95% of the mortality. The first manifestation of lung disease is often acough, followed by progressive dyspnea. Tenacious sputum becomespurulent due to colonization of bacteria. Chronic bronchitis andbronchiectasis can be partially treated with the current therapy, butthe course is punctuated by increasingly frequent exacerbations of thepulmonary disease. As the disease progresses, the patient's activity isprogressively limited. End-stage lung disease is heralded by increasinghypoxemia, pulmonary hypertension, and cor pulmonale.

The upper airways of the nose and sinuses are also involved by CF. Mostpatients develop chronic sinusitis. Nasal polyps occur in 15-20% ofpatients and are common by the second decade of life. Gastrointestinalproblems are also frequent in CF; infants may suffer meconium ileus.Exocrine pancreatic insufficiency, which produces symptoms ofmalabsorption, is present in the large majority of patients with CF.Males are almost uniformly infertile and fertility is decreased infemales.

Based on both genetic and molecular analyses, a gene associated with CFwas isolated as part of 21 individual cDNA clones and its proteinproduct predicted (Kerem, B. S. et al. (1989) Science 245:1073-1080;Riordan, J. R. et al. (1989)Science 245:1066-1073; Rommens, J. M. et al.(1989) Science 245:1059-1065)). European patent application publicationnumber: 0 446 017 A1 describes the construction of the gene into acontinuous strand, expression of the gene as a functional protein andconfirmation that mutations of the gene are responsible for CF. (Seealso Gregory, R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al.(1990) Nature 347:358-362).

The protein product of the CF associated gene is called the cysticfibrosis transmembrane conductance regulator (CFTR) (Riordan, J. R. etal. (1989) Science 245:1066-1073). CFTR is a protein of approximately1480 amino acids made up of two repeated elements, each comprising sixtransmembrane segments and a nucleotide binding domain. The two repeatsare separated by a large, polar, so-called R-domain containing multiplepotential phosphorylation sites. Based on its predicted domainstructure, CFTR is a member of a class of related proteins whichincludes the multi-drug resistance (MDR) or P-glycoprotein, bovineadenyl cyclase, the yeast STE6 protein as well as several bacterialamino acid transport proteins (Riordan, J. R. et al. (1989) Science245:1066-1073; Hyde, S. C. et al. (1990) Nature 346:362-365). Proteinsin this group, characteristically, are involved in pumping moleculesinto or out of cells.

CFTR has been postulated to regulate the outward flow of anions fromepithelial cells in response to phosphorylation by cyclic AMP-dependentprotein kinase or protein kinase C (Riordan, J. R. et al. (1989) Science245:1066-1073; Frizzell, R. A. et al. (1986) Science 233:558-560; Welsh,M. J. and Liedtke, C. M. (1986) Nature 322:467; Li, M. et al. (1988)Nature 331:358-360; Hwang, T-C. et al. (1989) Science 244:1351-1353;Anderson, M. P. and Welsh, M. J. (1992) Science 257:1701-1704.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). Population studies have indicatedthat the most common CF mutation, a deletion of the 3 nucleotides thatencode phenylalanine at position 508 of the CFTR amino acid sequence(ΔF508), is associated with approximately 70% of the cases of cysticfibrosis. This mutation results in the failure of an epithelial cellchloride channel to respond to cAMP (Welsh and Smith (1993) Cell73:1251-1254). In airway cells, this leads to an imbalance in ion andfluid transport. It is widely believed that this causes abnormal mucussecretion, and ultimately results in pulmonary infection and epithelialcell damage. In addition to the processing defect, the function ofCFTR-ΔF508 is decreased as indicated by a reduced P_(O) (Dalemans, W. etal. (1991) Nature 354:526-528; Denning, G. M. et al. (1992) Nature358:761-764). G551S, a mutation in NBD1, is correctly processed but hasaltered ATP-dependent channel regulation resulting in a reduced P_(O)(Anderson, M. P. and Welsh, M. J. (1992) Science 257:1701-1704). R117H,which contains a mutation in the membrane-spanning domain, is alsocorrectly processed, but has altered ion conducting properties producingan overall decrease in function (Sheppard, D. N. et al. (1993) Nature362:160-164).

To date, the primary objectives of treatment for CF have been to controlinfection, promote mucus clearance, and improve nutrition (Welsh M. J.et al. in The Metabolic Basis of Inherited Diseases, Vol. III, pp.3799-3876 (Scriver, C. R. et al. eds., McGraw-Hill, New York (1995)).Intensive antibiotic use and a program of postural drainage with chestpercussion are the mainstays of therapy. However, as the diseaseprogresses, frequent hospitalizations are required. Nutritional regimensinclude pancreatic enzymes and fat-soluble vitamins. Bronchodilators areused at times. Corticosteroids have been used to reduce intimation, butthey may produce significant adverse effects and their benefits are notcertain. In extreme cases, lung transplantation is sometimes attempted(Marshall, S. et al. (1990) Chest 98:1488).

Most efforts to develop new therapies for CF have focused on thepulmonary complications. Because CF mucus consists of a highconcentration of DNA, derived from lysed neutrophils, one approach hasbeen to develop recombinant human DNase (Shak, S. et al. (1990) Proc.Natl. Sci. Acad USA 87:9188). Preliminary reports suggest thataerosolized enzyme may be effective in reducing the viscosity of mucus.This could be helpful in clearing the airways of obstruction and perhapsin reducing infections. In an attempt to limit damage caused by anexcess of neutrophil derived elastase, protease inhibitors have beentested. For example, alpha-1-antitrypsin purified from human plasma hasbeen aerosolized to deliver enzyme activity to lungs of CF patients(McElvaney, N. et al. (1991 ) The Lancet 337:392). Another approachwould be the use of agents to inhibit the action of oxidants derivedfrom neutrophils. Although biochemical parameters have been successfullymeasured, the long term beneficial effects of these treatments have notbeen established.

Based on knowledge of the cystic fibrosis gene, three general correctiveapproaches (as opposed to therapies aimed at ameliorating the symptoms)are currently being pursued to reverse the abnormally decreased chloridesecretion and increased sodium absorption in CF airways. Defectiveelectrolyte transport by airway epithelia is thought to alter thecomposition of the respiratory secretions and mucus (Welsh M. J. et al.in The Metabolic Basis of inherited Diseases, Vol. III, pp. 3799-3876(Scriver, C. R. et al. eds., McGraw-Hill, New York (1995); Quinton, P.M. (1990) FASEB J. 4:2709-2717). Hence, pharmacological treatments aimedat correcting the abnormalities in electrolyte transport are beingpursued. Trials are in progress with aerosolized versions of the drugamiloride; a diuretic that inhibits sodium channels, thereby inhibitingsodium absorption. Initial results indicate that the drug is safe andsuggest a slight change in the rate of disease progression, as measuredby lung function tests (Knowles, M. et al. (1990) N. Eng. J. Med.322:1189-1194; App, E. (1990) Am. Rev. Respir. Dis. 141-605.)Nucleotides, such as ATP or UTP, stimulate purinergic receptors in theairway epithelium. As a result, they open a class of chloride channelthat is different from CFTR chloride channels. In vitro studies indicatethat ATP and UTP can stimulate chloride secretion (Knowles, M. et al.(1991) N. Eng. J. Med. 325-533). Preliminary trials to test the abilityof nucleotides to stimulate secretion in vivo, and thereby correct theelectrolyte transport abnormalities are underway.

As with all pharmacological agents, issues such as drug toxicity anddosing will be important in developing an appropriate pharmacologicalagent for treating CF. A more fundamental consideration withpharmacological approaches to CF therapy is whether the chloride channelactivity associated with CFTR is the crucial property that leads to thedisease state. The CFTR is an epithelial Cl⁻ channel with novelstructure and regulation (Welsh, M. J. et al. (1992) Neuron 8:821-829;Riordan, J. R. (1993) Annu. Rev. Physiol. 55:609-630). CFTR is composedof two membrane spanning domains which contribute to formation of theion conducting pore and three cytoplasmic domains that regulate channelactivity: Two nucleotide binding domains (NBDs), and the R domain. Thepresence of two NBDs confer a complex and poorly understood mechanism ofregulation on channel activity. Phosphorylation of the R domain bycAMP-dependent protein kinase (PKA) is necessary, but not sufficient,for channel activity. Once the R domain has been phosphorylated, theNBDs must bind (Anderson, M. P. and Welsh, M. J. (1992) Science257:1701-1704; Thomas, P. J. et al. (1992) J. Biol. Chem. 267:5727-5730;Ko, Y. H. et al. (1994) J. Biol. Chem. 269:14584-14588; Hartman, J. etal. (1992) J. Biol. Chem. 267:6455-6458; Travis, S. M. et al. (1993) J.Biol. Chem. 268: 15336-15339) and probably hydrolyze (Anderson, M. P.and Welsh, M. J. (1992) Science 257:1701-1704; Hwang, T. C. et al.(1994) PNAS USA 91:4698-4702; Baukrowitz, T. et al. (1994) Neuron12:473-482; Nagel, G. et al. (1992) Neuron 360:81-84) ATP in order toopen. In addition, ATP hydrolysis may be required to close the channel(Hwang, T. C. et al. (1994) PNAS USA 91:4698-4702; Baukrowitz, T. et al.(1994) Neuron 12:473-482). Studies of the CFTR containing site-directedmutations suggest that the two NBDs do not have equivalent functions inchannel regulation (Anderson, M. P. and Welsh, M. J. (1992) Science257:1701-1704), and it has been proposed previously that hydrolysis ofATP at NBD1 opens the channel, while hydrolysis of ATP at NBD2 regulatesclosure. An important goal of CF research is to understand the functionof CFTR and to use that knowledge to develop better treatments for thedisease.

A second approach to curing cystic fibrosis, "protein replacement" seeksto deliver functional, recombinant CFTR to CF mutant cells to directlyaugment the missing CFTR activity. The concept of protein replacementtherapy for CF is simple: a preparation of highly purified recombinantCFTR formulated in some fusogenic liposome or reassembled virus carrierdelivered to the airways by instillation or aerosol. However, attemptsat formulating a CF protein replacement therapeutic have met withdifficulties. For example, CFTR is not a soluble protein of the typethat has been used for previous protein replacement therapies or forother therapeutic uses. There may be a limit to the amount of a membraneprotein with biochemical activity that can be expressed in a recombinantcell. There are reports in the literature of 10⁵ -10⁶ molecules/cellrepresenting the upper limit (Wang, H. Y. et. al. (1989) J. Biol. Chem.264:14424), compared to 2000 molecules/second/cell being reported forsecreted proteins such as EPO, insulin, growth hormone, and tPA.

In addition to limited expression capabilities, the purification ofCFTR, a membrane bound protein, is more difficult than purification of asoluble protein. Membrane proteins require solubilization in detergents.However, purification of CFTR in the presence of detergents represent aless efficient process than the purification process required of solubleproteins. Other potential obstacles to a protein replacement approachinclude: 1) the inaccessibility of airway epithelium caused by mucusbuild-up and the hostile nature of the environment in CF airways; 2)potential immunogenicity; and 3) the fusion of CFTR with recipient cellsmay be inefficient.

A third approach to cystic fibrosis treatment is a gene therapy approachin which DNA encoding CFTR is transferred to CF defective cells (e.g. ofthe respiratory tract). However, methods to introduce DNA into cells aregenerally inefficient. Since viruses have evolved very efficient meansto introduce their nucleic acid into cells, many approaches to genetherapy make use of engineered defective viruses. However, viral vectorshave limited space for accommodating foreign genes. For example,adeno-associated virus (AAV) although an attractive gene therapy vectorin many respects, has only 4.5 Kb available for exogenous DNA. DNAencoding the full length CFTR gene represents the upper limit. Genetherapy approaches to CF will face many of the same clinical challengesas protein therapy.

Although there has been notable progress in developing curativetherapies for CF based on knowledge of the gene encoding CFTR, theexpressed protein product and mechanism of action, there are obstaclesconfronting every approach. New approaches for treating CF and otherdiseases or conditions associated inadequate or inappropriate functionof ATP-binding cassette (ABC) proteins are needed.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that inorganicpyrophosphates or their analogs can alter the activity of ATP-bindingcassette (ABC) proteins. As a result of this finding, the instantinvention provides new compositions and therapies for treating diseasesor conditions associated with inappropriate or inadequate activity ofABC proteins, particularly mammalian ABC proteins, such as the cysticfibrosis transmembrane conductance regulator protein (CFTR),P-glycoprotein, MHC-linked transport protein or peroxisomal membraneprotein.

In one aspect, the instant invention features pharmaceuticalcompositions of inorganic pyrophosphate (PPi) in a form that renders thePPi accessible to ABC proteins when administered to a subject in vivo.In preferred embodiments, the pharmaceutical composition of PPi includea delivery vehicle (e.g. a liposome, virosome or microsome) or ischemically modified to increase hydrophobicity and allow partitioningthough the lipid membrane of cells. The PPi can be in a form which ishydrolyzable or non-hydrolyzable (e.g., etidronate disodium,immidophosphate (PNP) or methylenediphosphonic acid (PCP)). In addition,the pharmaceutical composition is preferably of a dose effective foraltering the activity of an ATP-binding cassette (ABC) protein.

In another aspect, the invention features methods for treating orpreventing a disease or condition associated with inappropriate orinadequate ABC protein activity in a subject by administering to thesubject an effective amount of an inorganic pyrophosphate. In apreferred embodiment, the subject is a mammal and the ABC protein isselected from the group consisting of: CFTR, P-glycoprotein, MHC-linkedtransport protein or peroxisomal membrane protein. In one embodiment,the invention features methods for treating Cystic Fibrosis in a patientcomprising administering to the patient an effective amount of aninorganic pyrophosphate.

Inorganic pyrophosphates are small molecules that can be taken orally.In addition, since in vivo administration of certain PPi compositionsfor treating tumor-induced bone disease is unassociated with sideeffects, the pharmaceuticals should be safe.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of inorganic pyrophosphate (PPi) onkinetically modeled rate constants. A. A linear three state model ofCFTR channel activity composed of two closed states (C₁ and C₂), oneopen state (O), and four rate constants (β₁, β₂, α₁, and α₂). Panels B-Eshow values of rate constants before and after addition of 5 mM PP_(i).Rate constants were derived as described in the following examples fromfour experiments in which the membrane patch contained only one activechannel, studied in the presence of 0.3 mM ATP and 75 nM PKA. Asterisksindicate p<0.05.

FIG. 2 is a schematic diagram presenting a model showing the control ofthe nucleotide binding domain (NBD) and adenosine triphosphate (ATP)over CFTR gating. Panel A shows the effect of ATP on the gating ofphosphorylated CFTR channels; panel B shows the effect of ATP plusPP_(i). In each panel events at the two NBDs and corresponding channelgating (far right in each panel) are indicated from top to bottom.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention is based on the surprising finding that theadministration of inorganic pyrophosphate or a non-hydrolyzableinorganic pyrophosphate analog can stimulate the activity of a mutant orwild-type CFTR Cl⁻ channel in the presence of ATP. Based on thisfinding, the invention features methods for treating or preventing adisease or condition associated with inappropriate or inadequate ABCprotein activity.

Pharmaceutical Compositions

As used herein, the term "inorganic pyrophosphate (PPi)" refers to aninorganic ion consisting of two phosphate groups joined by aphosphate-phosphate high energy bond. The term is intended to includehydrolyzable inorganic pyrophosphates and non-hydrolyzable analogs.Examples of non-hydrolyzable inorganic pyrophosphate compounds includeetidronate disodium, immidophosphate (PNP) or methylenediphosphonic acid(PCP), which are all commercially available.

Certain non-hydrolyzable inorganic pyrophosphate compounds are currentlyadministered to treat tumor-induced bone disease (Fleich et al. (1991)Drugs 42(6):919-944). Although the effective dose is still a matter ofdebate, one slow infusion of 600 mg of PCP or 500 mg/day etidronate for3 days have shown efficacy. Administrations are to be repeated ifcalcaemia does not decrease satisfactorily after a few days or when theeffect subsides and blood calcium starts to rise again. It has also beenreported that calcaemia can be maintained after initial treatment by adaily oral administration of 1600 mg PCP.

Because ABC transporter proteins are membrane proteins, preferable PPipharmaceutical compositions are in a form that facilitates contact withand/or uptake by cell membranes. For example, PPi compounds can bechemically modified to increase their hydrophobicity and allowpartitioning though the lipid membrane of cells. For example, a chemicalmodification that is routinely used is an addition of phosphoesters tothe charged oxygens of the inorganic pyrophosphate. This modificationneutralizes the charge of the inorganic pyrophosphate and increases itshydrophobicity. By increasing the size of the phosphoester used (e.g.,methyl, ethyl or t-butyl esters), the level of hydrophobicity can beincreased. Phosphoester groups are easily hydrolyzed once they enter thecell, so that a phosphoester modification should not interfere with theability of inorganic pyrophosphates to bind to ABC transporter proteins.

Alternatively, inorganic pyrophosphate compositions can be administeredin conjunction with a delivery vehicle. As used herein, the term"delivery vehicle" refers to any hydrophobic moiety, which is useful fortransferring hydrophilic inorganic pyrophosphates across the lipidmembrane of a cell and into the cell cytosol where they can interactwith ABC proteins. Examples of appropriate hydrophobic moieties for usein the invention include detergent or other amphipathic moleculemicelles, membrane vesicles, liposomes, virosomes, and microsomes.Preferred hydrophobic moieties are naturally fusogenic or can beengineered to become fusogenic (e.g., by destabilizing lipid bilayers orendocytosis). Fusion proteins can be obtained from viruses such asparainfluenza viruses 1-3, respiratory syncytial virus (P-SV), influenzaA, Sendai virus, and togavirus fusion protein. Nonviral fusion proteinsinclude normal cellular proteins that mediate cell-cell fusion. Othernonvital fusion proteins include the sperm protein PH-30 which is anintegral membrane protein located on the surface of sperm cells that isbelieved to mediate fusion between the sperm and the egg. (See e.g.,Blobel et al. (1992) Nature 356:248-251). Still other nonviral fusionproteins include chimaeric PH-30 proteins such as PH-30 and the bindingcomponent of hemaglutinin from influenza virus and PH-30 and adisintegrin (e.g. bitistatin, barbourin, kistrin, and echistatin). Inaddition, lipid membranes can be fused using traditional chemicalfusogens such as polyethylene glycol (PEG).

Inorganic pyrophosphate compounds alone or optionally in conjunctionwith a delivery vehicle can be admixed with a pharmaceuticallyacceptable carrier and administered to a subject in need. A"pharmaceutically acceptable carrier" as used herein refers to amaterial that can be co-administered with an inorganic pyrophosphate andwhich allows the inorganic pyrophosphate to perform its intendedfunction (e.g., alter the activity of ABC proteins). Examples of suchcarriers include solvents, dispersion media, delay agents. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Any conventional media and agent compatible with theinorganic pyrophosphate can serve as a pharmaceutically acceptablecarrier for use in the subject invention.

Therapeutic Uses of the Pharmaceutical Compositions

CFTR-Based Diseases or Conditions

As described in further detail in the following Examples, inorganicpyrophosphates (PPis) have been shown to stimulate mutant CFTR Cl⁻channels by 2 or 3 fold, as evidenced by an increase in ion (e.g.chloride ion) transport across cellular membranes. PPi stimulates mutantCFTR Cl⁻ channel by binding to the nucleotide binding domain (NBD) inthe presence of ATP. This stimulation induces a significant change innet function, thereby alleviating the severity of the disease.Hydrolysis of PPi is not required for stimulation since hydrolyzable andnon-hydrolyzable forms of PPi were equally effective.

Present data suggest that very little CFTR function is required toprevent disease or attenuate its severity. Individuals who have only8-10% of normal CFTR transcripts maintain normal airways function (Chu,C. S. et al. (1992) J. Clin. Invest. 90:785-790). In addition, CFTRmutants which retain partial Cl⁻ channel activity are associated with amilder clinical phenotype (Sheppard, D. N. et al. (1993) Nature362:160-164). Individuals with residual agonist-induced Cl⁻ secretiontend to have milder clinical symptoms and a later age at diagnosis(Veeze, H. J. et al. (1994) J. Clin. Invest. 93:461-466). Thus if PP_(i)increased the activity of mutant CFTR by 2 or 3 fold (as shown in theExample below), it is possible that it could produce a significantchange in net function and might alter the severity of the disease.

It is possible that interaction of PPi with the NBD of CFTR improves theprocessing of mutant CFTR. For example, it is known that CFTR ΔF508 hasdefective processing. An interaction with PPi might change theconformation or the volting and thereby allow the mutant ΔF508 proteinto escape from the endoplasmic reticulum and be delivered to the plasmamembrane.

In addition, the data presented in the following Example providesevidence that PPi can stimulate wild-type (i.e. non-mutant) CFTR. CFTRis known to be important in fluid and electrolyte secretion by theintestine. Therefore stimulation of CFTR by PPi could be useful fornon-CF patients, who suffer from constipation or other electrolyte orfluid disorders.

Since PPi has been found to bind to CFTR's nucleotide binding domains(NBD) and NBDs are highly conserved among the ABC family oftransporters, PPi should interact with other ABC proteins, particularlyother mammalian ABC proteins, such as PMP70s, MHC-linked (TAP) transportproteins, and P-glycoproteins, potentially resulting in a therapeuticeffect.

PMP70-Based Diseases or Conditions

Peroxisomes are cytoplasmic organelles containing enzymes for theproduction and decomposition of hydrogen peroxide. Peroxisomes arethought to be involved in oxidative processes in vivo and dysfunctionalperoxisomes are thought to be involved in infantile Refsum disease,hyperpipecolic acidaemia and neonatal adrenoleukodystrophy.

Peroxisomes are morphologically absent in cells of patients withZellweger syndrome (Goldfischer, S. et al. (1973) Science 182:62-64).Biogenesis of peroxisomes is apparently impaired in this disorder causedby defective protein machinery (Santos, M. J. et al. (1988) Science239:1536-1538). It has been further postulated that the primarybiochemical lesion is at the level of the biosynthesis of a proteinessential for the import of peroxisomal enzymes from the cytoplasm intothe peroxisomes which utilizes adenosine triphosphate during import(Schutgens et al. (1986) Eur. J. Pediatr. 144:430-440; Imanaka, T.(1987) J. Cell Biol. 105:2915). If the lesion resulting in Zellweger'sSyndrome or in another peroxisome-based disease is in the biosynthesisof the PMP70 protein, pharmaceutical compositions of the presentinvention would be useful in potentiating the activity of existing PMP70proteins, so that they can compensate for the reduced synthesis and thusalleviate disease phenotypes.

MHC-linked (TAP) transport protein-Based Diseases or Conditions

TAP proteins are involved with transporting peptides into theendoplasmic reticulum of cells where they interact with Class Imolecules. TAP transporters are therefore important in antigenpresentation. The work described in the following Example suggests thatnon-hydrolyzable PPi compositions will interact with the NBD ofMHC-linked (TAP) transport proteins, preventing hydrolysis and therebyinhibiting function.

The interaction of non-hydrolyzable PPi analogs with a TAP transportprotein, blocking antigen presentation by the human leukocyte antigen(HLA) system suggests a number of therapeutic uses. For example, in vivoadministration of PPi should be of use in treating an autoimmunedisease, by preventing presentation of antigens that are the basis ofautoimmunity. Furthermore, administration of inorganic pyrophosphateanalogs could be of value in prevention of organ transplant rejection.For example, the transplanted organ may present antigen to thepostimmune system thereby producing rejection. Inhibition of TAPtransporters via administration of inorganic pyrophosphate analogs couldprevent or ameliorate this effect. In addition, the binding ofnon-hydrolyzable inorganic pyrophosphates to TAP transporters canminimize recognition of any foreign protein by the host immune system.For example, non-hydrolyzable PPi could be administered in conjunctionwith gene therapy vectors to prevent an immune response to the vectorsby a subject.

P-glycoprotein-based diseases or conditions

Analogous to bacterial ABC transporters, P-glycoprotein has beenpostulated to function as an energy-requiring drug-efflux pump.P-glycoprotein is also thought to be involved with nutrient absorption,particularly from the intestine. Overexpression of P-glycoproteinconfers the phenotype of multidrug resistance (mdr), which may causechemotherapies (e.g. against cancer or infection) to fall. It is thoughtthat P-glycoprotein binds the drugs to which a mdr cell is resistant orcollaterally sensitive.

The binding of non-hydrolyzable pyrophosphate compounds to the NBD ofP-glycoproteins should inhibit the activity of P-glycoprotein bypreventing hydrolysis. As a result it would useful to adminsternon-hydrolyzable pyrophosphate compounds in conjunction withchemotherapies.

For therapy, a PPi pharmaceutical composition can be administered to a"subject", which is preferably a mammal (e.g., a mammal such as a human,dog, cat, horse, cow, goat, rat or mouse), by any route that allows theinorganic pyrophosphate to perform its intended function. Examples ofroutes of administration which can be used include injection(subcutaneous, intravenous, parenterally, intraperitoneally, etc.),oral, inhalation (via an aerosol), transdermal, and rectal. A preferredroute of administration is by inhalation (e.g., of an aerosolizedpharmaceutical composition). In addition, prior to administration, itmay be useful to administer agents to clear obstructions to targetcells. For example, it may be usefuI to remove mucus (e.g. byadministering DNAse) prior to administering an aerosolized PPipharmaceutical composition to the lungs of a CF patient.

Depending on the route of administration, the lipid vesicle can becoated with or in a material to protect it from the natural conditionswhich may detrimentally effect its ability to perform its intendedfunction. Dosage regimes may be adjusted for purposes of improving thetherapeutic response to the membrane-associated protein of the lipidvesicle. For example, several divided doses may be administered daily orthe dose may be proportionally reduced as indicated by the exigencies ofthe therapeutic situation.

An "effective amount" of inorganic pyrophosphate is intended to includethat amount sufficient or necessary to significantly reduce or eliminatea subject's symptoms associated with CF. The amount can be determinedbased on such factors as the type and severity of symptoms beingtreated, the weight and/or age of the subject, the previous medicalhistory of the subject, the selected route for administration of theagent, and the type of CF mutation. Over 200 different varieties of CFmutation have been identified to date, e.g., ΔF508, R117H, or G551S (seefor example Tsui, L-C (1992) "The Spectrum of Cystic Fibrosis Mutations"Trends in Genetics 8 (11) 329-398), some are associated with more severesymptoms than others. The determination of appropriate "effectiveamounts" is within the ordinary skill of the art.

The present invention is further illustrated by the following examplewhich in no way should be construed as limiting the invention. Thecontents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Example The Effect of PPi and PPi Analogs on the CFTR Cl⁻ ChannelActivity Materials and Methods

(i) Chemicals and Solutions

Sodium pyrophosphate (PPi) was obtained from EM Science, Gibbstown, N.J.Catalytic subunit of PKA was from Promega Corp., Madison Wis.Methylenediphosphonic acid (PCP, trisodium salt tetrahydrate) wasobtained from Aldrich Chemical Co., Milwaukee, Wis. Etidronate disodium(1-hydroxyethylidenebisphosphonic acid, Didronel, 300 mg/6 ml H₂ O), wasobtained from Pharma, Minneapolis, Minn. Adenosine 5'-triphosphate (ATP;disodium salt), imidodiphosphate (PNP, sodium salt), and all otherreagents were obtained from Sigma Chemical Co., St. Louis, Mo.

For experiments with excised inside-out membrane patches, the pipette(extracellular) solution contained (in mM): 140 NMDG(N-methyl-D-glucamine), 100 aspartic acid, 35.5 HCl, S CaCl2, 2 MgCl2,10 HEPES (4- 2-hydroxyethyl!-1-piperazine ethanesulfonic acid), pH 7.3with 1N NaOH. bath (intracellular) solution contained (in mM): 140 NMDG,135.5 HCl, 3 MgCl2, 10 HEPES, 4 Cs and 1 EGTA, pH 7.3 with 1N HCl (Ca2+!free<10-8M). PPi and PPi analog stock solutions were 200 mM inCsEGTA-free bath solution, pH 7.3, and diluted to desired finalconcentration in bath solution, except for etidronate disodium, whichwas diluted from commercial preparation. For Ussing chamber experiments,the mucosal (apical) solution contained (in mM): 135 NACl, 1.2 MgCl2,1.2 CaCl2, 2.4 K2HPO4, 0.6 KH2PO4, and 10 HEPES, pH 7.3. The submucosal(basolateral) solution contained (in mM): 135 Na gluconate, 7 mM MgSO4,2.4 K2HPO4, 0.6 KH2PO4, 10 HEPES, 10 dextrose, no added calcium, and 1MgATP, pH 7.3.

(ii) Cells and Transaction Procedure

For patch clamp experiments, three different cell types expressingwild-type and mutant CFTR were used: stably transfected C127 mousemammary epithelia cells, NIH 3T3 fibroblasts, or transiently transfectedHeLa cells. Transient transfection of HeLa cells with the vacciniavirus/bacteriophage T7 hybrid expression system was as previouslydescribed (Fuerst, T. R. et al. (1986) PNAS USA 83:8122-8126; Carson, M.R. and Welsh, M. J. (1993) Am. J. Physiol. 265:L27-L3). Similar resultswere obtained with all cell types, and the data are combined. Forexperiments with ΔF508 CFTR, stably transfected cells were incubated at25 C. for 12-72 h prior to use (Denning, G. M. et al. (1992) Nature358;761-764). For experiment with polarized T-84 intestinal epithelialmonolayers, cells were plated at a density of 5×10⁵ cells/cm² onpermeable filter supports (Millicell HA filters, Millipore, Bedford,Mass., 0.4 μm pore size, 27 mM diameter). Transepithelial resistance wasmonitored with a EVOM epithelial voltohmmeter (World PrecisionInstruments, Sarasota, Fla.), and filters with resistances between 4 to7 kΩ were used.

(iii) Patch-clamp technique

Methods for excised, inside-out patch-clamp recording are similar tothose previously described (Carson, M. R. and Welsh, M. J. (1993) Am. J.Physiol. 265:L27-L32; Hamill, O. P. et al. (1981) Pfluegers Arch.391;85-100; Anderson, M. P. et al. (1991) Cell 67:775-784). An Axopatch200 amplifier (Axon Instruments, Inc., Foster City), was used forvoltage-clamping and current amplification. A microcomputer and thepClamp software package (Axon Instruments, Inc.) were used for dataacquisition and analysis. Data were recorded on videotape followingpulse code modulation using a PCM-2 AID VCR adapter (Medical SystemsCorporation, Greenvale, N.Y.). Patch pipettes were fabricated asdescribed (Carson, M. R. and Welsh, M. J. (1993) Am. J. Physiol.265:L27-L32), with pipette resistances of 5 to 15 MΩ, and with sealresistance routinely greater than 5 GΩ. Voltages are referenced to theextracellular side of the membrane. Excised macro-patch experiments wereperformed at a holding potential of -40 or -80 mV; single channel datawere recorded at a holding potential of -80 mV. Experiments wereconducted at 34°-36° C. using a temperature-controlled microscope stage(Brook Industries, Lake Villa, Ill.).

For excised macro-patch data, replayed records were filtered at 1 kHzusing a variable 8-pole Bessel filter (Frequency Devices Inc.,Haverhill, Mass.) and digitized at 2 kHz. Each time course data pointrepresents the average current from 1 s with one data point collectedevery 5 s. Average currents for an intervention were determined as theaverage of the last 12 data points (the last rain) during theintervention. To compensate for any channel run-down during anexperiment, specific interventions were bracketed when possible withcurrent measurements made with similar concentrations of ATP but withoutthe test compound; the intervention current was then compared to theaverage of pre- and post-intervention currents. For single channelanalysis, replayed data were filtered at 1 kHz using a variable 8-poleBessel filter, digitized at 5 kHz, and digitally filtered at 500 Hz.Idealized records were created using a half-height transition protocol;transitions less than 1 ms in duration were not included in theanalysis. For the purpose of illustration, time-course figures areinverted so that an upward deflection represents an inward current, datapoints during solution perfusion were not included in some figures, andsingle channel traces were digitally filtered at 200 Hz.

Burst analysis was performed as previously described (Carson, M. R. etal. (1994) Biophys. J. (In Press)), using a t_(c) (the time whichseparates interburst closures from intraburst closures) of 20 ms. Thisvalue was derived from analysis of wild-type CFTR closed-time histogramsderived from excised inside-out membrane patches containing a singlechannel studied in the presence of 1 mM ATP plus PKA, and by the methodof Sigurdson et al. (Sigurdson, W. J. et al. (1987) J. Exp. Biol.127:191-209). Closures longer than 20 ms were considered to defineinterburst closures, while closures shorter than this time wereconsidered gaps within bursts. Burst data for PPi, PNP, and PCP werederived from experiments in which the membrane patch contained oneactive channel. For experiments with etidronate and ΔF508 CFTR, burstdata were from patches containing four or fewer active channels; burstsin which there were no superimposed openings and which were separatedfrom other bursts by greater than 20 ms were included in the analysis.It has been previously shown that no discernible bias is observed byincluding burst data from patches with more than one channel.

(iv) Maximum likelihood analysis and kinetic modeling

Maximum likelihood analysis and kinetic modeling were performed asdescribed (Winter, M. C. et al. (1994) Biophys. J. 66:1398-1403).Briefly, unfiltered single channel data were digitized on amicrocomputer (Apple Macintosh, Apple Computer, Inc., Cupertine, Calif.)equipped with a multifunctional data acquisition board (NB-MIO-16) andLabVIEW 2 software (National Instruments, Austin, Tex.) at 5 kHzfollowing filtering with an 8-pole Bessel filter (Frequency DevicesInc., Haverhill, Mass.) at a corner frequency of 1 kHz with subsequentdigital filtering at 500 Hz. Previous work has shown that a linear threestate model (C₁ ⃡C₂ ⃡O) best describes the kinetics of CFTR channelactivity (Winter, M. C. et al. (1994) Biophys. J. 66:1398-1403). TheMaple 5 symbolic algebra program (Waterloo Maple Software, Waterloo,Canada) was used to derive the open and closed time probability densityfunctions for this model by solving the matrix equations in terms of therate constants. The resulting equations were used in LabVIEW 2 todetermine the set of rate constants which yielded the maximum likelihoodfor the open and closed times observed with different experimentalinterventions.

(v) α-³² P!8-N3ATP Photolabeling

Photolabeling of membrane-associated CFTR was performed as previouslydescribed (Travis, S. M. et al. (1993) J. Biol. Chem. 268:15336-15339).Monolayer cultures of Spodoptera frugiperda (Sf9) cells were infectedwith a baculovirus containing the entire coding sequence for human CFTR(gift of R. J. Gregory and A. E. Smith, Genzyme Corp., Framingham,Mass.). Membranes were prepared by differential centrifugation andresuspended in 20 mM HEPES, pH 7.5, 50 mM NaCl, 3 mM MgSO4, with 2 μg/mleach of leupeptin, aprotinin, and pepstatin. Membrane-associated CFTRwas phosphorylated prior to photolabeling by incubation with 100 nM PKAand 0.1 mM ATP in buffer containing 10 m MgCl2 and 50 mM 1,4-piperazinediethane sulfonic acid, pH 6.8 for 20 min at 30° C., then diluted in 12volumes buffer containing 20 mM tris(hydroxymethyl)aminomethane, pH 7.5,0.2 mM EGTA and 1 μM calyculin A. Membranes were collected bycentrifugation and resuspended at a concentration of 2.5 mg protein/mlin the same buffer.

Photolabeling was performed by preincubating membranes (50 μg membraneprotein/sample) on ice with α-³² P!8-N3ATP (30 μM, 6-12 Ci/mmol) and PPi(in mM). After 60 s UV irradiation, CFTR was solubilized andimmunoprecipitated as described (Travis, S. M. et al. (1993) J. Biol.Chem. 268:15336-15339) using antibodies raised against the R domain(M13-1, 0.3 μg/sample) and against the C terminus (M1-4, 10 μg/sample).Immunocomplexes were analyzed by SDS-PAGE and incorporation of α-³²P!8-N3ATP was quantitated with an AMBIS radioanalytic imaging system(AMBIS Systems, Inc., San Diego, Calif.). Data are expressed as percentof radiolabel incorporation relative to control which had no added PPi.

(vi) Measurement of apical membrane Cl⁻ current

Apical membrane Cl⁻ current was measured as previously described(Ostedgaard, L. S. et al. (1992) Am. J. Physiol. 263:L104-L112). T-84monolayers with resistances between 4-7 kΩ were mounted in modifiedUssing chambers (Jim's Instruments, Iowa City, Iowa), and thebasolateral membrane was permeabilized by addition of approximately 100μg/ml S. aureus α-toxin to the serosal solution. S. aureus α-toxinproduces pores in the basolateral membrane which are large enough toallow passage of ions and small molecules such as nucleotides, withoutpermitting exchange of larger proteins and cellular constituents(Ostedgaard, L. S. et al. (1992) Am. J. Physiol. 263 :L104-L112; Fussle,R. et al. (1981) J. Cell Biol. 91:83-94). Permeabilization was confirmedby both a transient current upon addition of toxin which returned tobaseline, as well as by stimulation of short circuit current by additionof 10 μM cAMP, which is cell impermeant in the absence ofpermeabilization. Data are expressed as current observed five min afterthe intervention.

Results are means ±SEM of n observations. Statistical significance wasassessed using a paired, unpaired, or one population Student's t-test asappropriate.

RESULTS

(i) Pyrophosphate stimulates CFTR Cl⁻ current

To determine if inorganic pyrophosphate (PP_(i)) would alter CFTR Cl⁻channel activity, 5 mM PP_(i) was added to an excised, inside-outmembrane patch containing many CFTR channels which had beenphosphorylated by PKA. It was shown that addition of 5 mM PP_(i) in thepresence of 0.3 mM ATP produced a reversible increase in Cl⁻ current.Addition of PP_(i) in the absence of ATP did not stimulate current,suggesting that PP_(i) alone cannot open CFTR Cl⁻ channels or substitutefor ATP in supporting activity. Furthermore, as the concentration ofPP_(i) increased, the stimulation increased with apparent EC₅₀ ofapproximately 500 μM. The stimulatory effect of PP_(i) was only observedwhen added to the cytosolic aspect of the membrane patch; externaladdition of 5 mM PP_(i) did not alter currents from cells studied in thewhole-cell configuration (n=4).

To determine how PP_(i) stimulated CFTR currents, membrane patches thatcontained only a single active channel were studied. When 5 mM PP_(i)was added to the cytosolic surface of the membrane patch in the presenceof 0.3 mM ATP and 75 nM PKA several traces were produced. Examination ofthe traces suggest four things. First, PP_(i) did not increase Cl⁻current by changing the amplitude of current flowing through a single cchannel. In eight experiments, current amplitude was 0.90±0.05 pA beforeand 0.91±0.02 pA after addition of PP_(i) (P=0.812). Second, it isapparent that the increase in total current is due to an increase in theprobability that single channels are in the open state (P_(O)). Third,it appears that the increased P_(O) is at least in part caused by anincrease in the duration of bursts of activity. (Note that a burst isdefined as the time in which the channel is open with only briefflickers to the closed state. Bursts are delimited by long closures ofgreater than 20 ms). Fourth, PP_(i) appeared to decrease the duration ofthe long closed states between bursts of activity. PP_(i) increasedP_(O) from 0.39+0.021 to 0.81+0.03 (n=8, p<0.001) and increased meanburst duration from 175+6 ms to 1568±219 ms (n=8, p<0.001).

To provide more insight into the mechanism by which PP_(i) stimulatedCFTR, kinetic modeling was performed on the data from four patches whichcontained only a single channel. It has been previously shown that theactivity of single phosphorylated CFTR channels can be described by alinear three state model (Winter, M. C. et al. (1994) Biophys. J.66:1398-1403), and this model has been used to describe how ATP, ADP,and inorganic phosphate alter the rate constants which describetransitions between states (Carson, M. R. et al., 1994 Biophys. J (InPress); Winter, M. C. et al., 1994 Biophys. J. 66:1398-1403). As shownin FIG. 1A, C₁ represents the long closed state between bursts ofopenings and C2⃡O represents the bursting state, in which several channelopenings (O) are separated by brief, flickery closures (C2), before thechannel returns to the longer closed state (C1). The rate constants (β₁,β₂, α₁, and α₂) describe the rate of transition between each state.

FIGS. 1B-1E describe the average values of the rate constant in thepresence and absence of 5 mM PP_(i) , PP_(i) produced large changes inboth β₁ and α₁, a smaller decrease in α₂, and did not alter β₂. Theseresults suggest that PP_(i) affects more than one step in channelgating. The increase in burst duration is caused principally by asix-fold decrease in α₁. α₁ is a major determinant of burst durationbecause it defines the rate at which the channel leaves the burstingmode (i.e. leaves C₂, the closed state within a burst). The duration ofbursts can also be affected by β₂ and α₂, the transition rates within aburst. PP_(i) did not alter β₂, but decreased α₂ by 37%. However,because α₂ is one tenth the magnitude of β₂, the decrease in α₂ producedless than a 5% change in P_(O) within a burst (n=4, not significantlydifferent). Thus the decrease in α2 had a minimal effect on net channelactivity.

In addition to increasing burst duration, PP_(i) also increased β₁, therate of transition from the long closed state (C₁) to the bursting state(C₂ ⃡0), suggesting that PP_(i) functioned as a channel opener.Interestingly, β1 is the only rate constant which is affected by ATP andADP (Winter, M. C. et al. (1994) Biophys. J. 66:1398-1403). In othersystems, PP_(i) binding to NBDs can mimic some aspects of ATP binding.These data suggest that PP_(i) may potentiate channel activity byinteracting with CFTR at the one or both NBD s and may produce effectssimilar to those that occur upon ATP binding.

Together, the single channel analysis and kinetic modeling data showthat in the presence of ATP, PP_(i) had two primary effects on CFTR:first, PP_(i) acted as a channel opener, and second, PP_(i) preventedchannel closure by prolonging the burst state.

(ii) Pyrophosphate increases 8-N₃ ATP binding

One effect of PP_(i) was to increase the rate at which channels opened.It has been previously suggested that ATP binding and hydrolysis arenecessary for the channel to open. Therefore the possibility that PP_(i)might alter the binding of ATP to CTFR was tested by examining theeffect of 5 mM PP_(i) on 8-N₃ ATP photolabeling of membrane-associatedCFTR in Sf9 cells. Previous studies showed that CFTR was labeled in aspecific and saturable way by 8-N₃ ATP, that 8-N₃ ATP supported channelfunction, and that ATP competed with 8-N₃ ATP (Travis, S. M. et al.(1993) J. Biol. Chem. 268:15336-15339), suggesting that 8-N₃ ATP bindingis an appropriate assay of the functional ATP binding sites in CFTR.

In these experiments, PP_(i) produced a concentration-dependent increasein 8-N₃ ATP labeling of CFTR, suggesting that PP_(i) alters nucleotidebinding kinetics. Since the photolabeling reaction was performed over a60 s period, increased labeling of CFTR by PP_(i) could be due to anincrease in the rate of nucleotide binding to, and/or a decrease in therate of nucleotide release from, one or both of the NBDs. Thus, PP_(i)appears to increase the amount of ATP bound to CFTR.

(iii) Nonhydrolyzable PP_(i) analogs stimulate CFTR channel activity

Because it has been proposed that hydrolysis of ATP is necessary forchannel activity (Anderson, M. P. and Welsh, M. J. (1992) Science257:1701-1704, Hwang, T-C. et al. (1994) Proc Natl Acad Sci91:4698-4702, Anderson, M. P. et al. (1991) Cell 67:775-784), whetherPP_(i) may stimulate channel activity was considered by providing anonhydrolyzable substrate. To determine if hydrolysis of PP_(i) wasnecessary for the observed increase in channel activity or burstduration, the effect of the nonhydrolyzable PP_(i) analogsimidodiphosphate (PNP), methylenediphosphonic acid (PCP), and etidronatedisodium, which is a drug used clinically for treatment ofhypercalcemia, was assessed (Fleisch, H., (1991) Drugs 42:919-944).Experiments were carried out in which the effects of PCP, PNP andetidronate were examined. Experiments were carried out in two separateexcised membrane patches from C127s cells which contained only a singleactive channel. For all interventions 75 nM PKA and 0.3 mM ATP werepresent on the cytosolic surface and the PP_(i) analogs were added at aconcentration of 5 mM. These experiments showed that like PP_(i), PCP,PNP, and etidronate prolonged bursts of activity. However, the prolongedbursts generated by these compounds were less frequent than wereobserved with PP_(i), suggesting that they were less potent.

The effects of these agents on P_(O) and average burst duration werealso studied. All three nonhydrolyzable analogs altered channelactivity. The effects of PCP, however, did not achieve statisticalsignificance, suggesting that small differences between compounds, suchas the electronegativity and/or angle of the bridging group, can producea large difference in the ability to stimulate the channel. This issimilar to the finding that AMP-PNP, a nonhydrolyzable ATP analog with astructure very similar to ATP (Yount, R. G., et al. (1971) Biochem10:2484-2489), has a binding affinity one twentieth that of ATP (Travis,S. M. et al. (1993) J. Biol. Chem. 268:15336-15339). Although these datado not rule out the possibility that hydrolysis of PP_(i) may occur,they suggest that hydrolysis of PP_(i) is not necessary for stimulationof the channel activity or prolongation of burst duration.

The ability of PP_(i) to potently stimulate CFTR channel activitysuggested that PP_(i) or a more stable nonhydrolyzable PP_(i) analogmight be a useful pharmacological agent to stimulate poorly functionalCFTR channels that are associated with disease. To support a possiblepharmacological approach, three criteria must be met. First, PP_(i) muststimulate CFTR channels which contain CF-associated mutations. Second,PP_(i) or an analog must be able to stimulate endogenous CFTR in theapical membrane of epithelia. And third, since stimulation occurs fromthe cytoplasmic aspect, PP_(i) or an analog must be able to gain accessto the interior of the cell. To address the first two issues, weperformed the following studies.

(iv) PP i stimulates CFTR containing CF-associated mutations

The effect of PP_(i) on CFTR containing the CF-associated mutationsΔF508, R117H, and G551S was examined. These mutations were studiedbecause they occur in different regions of the protein and havedifferent mechanisms of dysfunction. CFTR-ΔF508, the most common CFcausing mutation (Cutting, G. R. et al. (1990) Nature 346:366-369;Kerem, B-S et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451), isdefectively processed and fails to traffic to the plasma membrane. Inaddition to the processing defect, the function of CFTR-ΔF508 isdecreased as indicated by a reduced P_(O) (Dalemans, W. et al. (1991)Nature 354:526-528; Sheppard, D. N. et al. (1993) Nature 362:160-164).G551S, a mutation in NBD1, is correctly processed but has alteredATP-dependent channel regulation resulting in a reduced P_(O) (Anderson,M. P. and Welsh, M. J. (1992) Science 257:1701-1704). R117H, whichcontains a mutation in the membrane-spanning domain, is also correctlyprocessed, but has altered ion conducting properties producing anoverall decrease in function (Sheppard, D. N. et al. (1993) Nature362:160-164).

Experiments were carried out in which a patch containing two CFTR-ΔF508channels was used to study effects of PP_(i) administration. In thepresence of 75 nM PKA and 0.3 mM ATP, channel openings appearqualitatively similar to those of wild-type CFTR, except that both P_(O)and burst duration are less than wild-type. Addition of 5 mM PP_(i)stimulated ΔF508 activity three-fold (n=5). P_(O) increased three-fold(from 0.07±0.01 to 0.21±0.022), and average burst duration increasedeight-fold (from 125±19 ms to 1023±330 ms) (n=3). A similar stimulationof channel activity was observed when 1 mM PP_(i) was added toCFTR-R117H and CFTR-G551S.

(v) Pyrophosphate stimulates apical membrane Cl⁻ currents

To learn whether PP_(i) can stimulate endogenous CFTR in the apicalmembrane of epithelia, the effect of PP_(i) was measured onpermeabilized T-84 intestinal epithelial monolayers. T-84 intestinalepithelial cells grow as a polarized epithelium on permeable supportsand express CFTR in their apical membrane (Madara, J. L. et al. (1987)Gastroenterology 92:1133-1145, Denning, G. M. et al. (1992) J. ClinInvest 89:339-349) Because PP_(i) is hydrophilic and membraneimpermeant, the basolateral membrane was permeabilized with S. aureusα-toxin (100 μg/ml) to provide access of small molecules, such as cAMPand PP_(i), to the cell interior (Ostedgaard, L. S. et al. (1992) Am. J.Physiol. 263:L104-L112). Basolateral solutions contained 1 mM ATP.Permeabilizing the basolateral membrane also allowed for current flow tobe measured across the apical membrane in the absence of the basolateralmembrane barrier.

After permeabilization, the baseline Cl⁻ current was small suggestingthat in the absence of cAMP, the apical membrane was relativelyimpermeable to C⁻. As previously reported, addition of cAMP to thebasolateral bathing solution stimulated Cl⁻ current (Ostedgaard, L. S.et al. (1992) Am. J. Physiol. 263:L104-L112). Subsequent addition ofeither 1 or 5 mM PP_(i) to the basolateral bathing solution produced afurther increase in current.

Although PP_(i) can function as a nonspecific phosphatase inhibitor, andCFTR channel activity is regulated in part by phosphorylation (Berger,H. A. et al. (1992) J. Biol Chem 268:2037-2047; Berger, H. A. et al.(1994) Jap J Phsiol (in press)), it is unlikely that PP_(i) stimulatedthe apical membrane Cl⁻ current by acting as a nonspecific phosphataseinhibitor because removal of cAMP in the continued presence of PP_(i)decreased current to baseline levels within 3 min. This rate is similarto that observed in the absence of PP_(i). If the effect of PP_(i) wasin part due to phosphatase inhibition, it would be expect for thecurrent to be maintained after removal of cAMP.

In other experiments, addition of 1 or 5 mM PP_(i) to permeabilizedmonolayers in the absence of cAMP did not increase current abovebaseline levels. This result suggests that PP_(i) only stimulates apicalCl⁻ current in the presence of cAMP, a result consistent with thefindings that PP_(i) only stimulated phosphorylated CFTR Cl⁻ channels inexcised membrane patches.

(vi) Mechanism of Action

Although not wishing to be bound by theory, the results obtained in theabove-described experiments support the following possible mechanism ofaction of PP_(i) or its nonhydrolyzable analogs on stimulation ofwild-type or mutant CFTR Cl⁻ channels.

PP_(i) binds to the glycine-rich loop in a number of proteins thatcontain nucleotide binding domains (Peinnequin, A. et al. (1992) Biochem31:2088-2092; Michel, L. et al. (1989) Biochem 28:10022-10028; Issartel,J. P., et al. (1987) J. Biol. Chem 262 28:13538-13544; Thomas, P. J. etal. (1992) J. Biol. Chem. 267:5727-5730; Barrels, E. M. et al. (1993)Biochim Biophys. Acta 1157:63-73; and Greene, L. E. et al. (1980) J BiolChem 255:543-548). Data described in Example I below show that PP_(i)also interacted with CFTR and reversibly stimulated the activity ofphosphorylated channels. PP_(i) was not able to stimulate activity onits own; it required the simultaneous presence of ATP. The ability ofnon-hydrolyzable analogs such as PNP, PCP, and etidronate to mimic theeffect of PP_(i) indicates that hydrolysis of PP_(i) is not required forstimulation.

The data show that PP_(i) increased the channel open probabilitypredominantly by affecting the rate of two transitions. PP_(i) increasedβ₁, the rate at which the channel leaves the long closed state andenters a burst of activity. PP_(i) also decreased α_(i), the rate atwhich the channel leaves a burst and returns to the long closed state.Although either an increase in β₁ or a decrease in α₁ will increaseP_(O), previous work indicates the β₁ and α₁ transitions are distinctsteps that are not directly related (Winter, M. C. et al. (1994) BiophysJ 66:1398-1403).

First, PP_(i) binding itself cannot open the channel because PP_(i) didnot support channel activity alone; the stimulatory effect of PP_(i)required the presence of ATP. This observation is consistent withprevious data suggesting that ATP binding and hydrolysis is required toopen the channel (Hwang, T.-C et al. (1994) Proc Natl Acad Sci91:4698-4702; Baukrowitz, T. et al. (1994) Neuron 12:473-482; andAnderson, M. P. et al. (1991) Cell 67:775-784). Second, the findingsthat PP_(i) stimulated a rate (β₁) that is controlled by ATP, and thatPP_(i) increased the binding of the ATP analog 8-N₃ ATP indicate thatthe effect of PP_(i) is allosteric. It was concluded that PP_(i) bindsto a site in CFTR other than the site at which an ATP interactiondirectly opens the channel. When PP_(i) binds to the allosteric site, itfacilitates binding (and perhaps hydrolysis) of ATP, increasing the rateat which the channel opens. These data are the first direct evidence fora compound having an allosteric effect on CFTR gating activity.

Previous work indicated that mutations expected to reduce the rate ofhydrolysis at NBD2 increased the duration of bursts. Non-hydrolyzableanalogs of ATP also prolonged the duration of bursts (Baukrowitz, T. etal. (1994) Neuron 12:473-482; Gunderson, K. L. et al. (1994) J. Biol.Chem 269:19349-19353) as does PP_(i) when added to CFTR studied inartificial lipid bilayers (Gunderson, K. L. et al. (1994) J. Biol. Chem269:19349-19353)). These data suggested that the rate of hydrolysis atNBD2 is a primary factor in terminating bursts and thus in determiningtheir duration. The data suggests that PP_(i) prolongs the duration ofbursts by binding to NBD2 which stabilizes the bursting state; becausePP_(i) is not hydrolyzed, the interaction is prolonged, the terminationof a burst is delayed, and α₁ decreases.

A model of how events at NBD1 and NBD2 give rise to the opening andclosing of phosphorylated channels is shown in FIG. 2A, where verticalcolumns represent events occurring at NBD1, at NBD2, and thecorresponding channel activity. A key feature of this model is that thetwo NBDs have different functions. In the absence of ATP the channel isclosed (top of FIG. 2A). Binding of ATP to NBD2 allosterically regulatesevents at NBD1, including a step leading to channel opening, which maybe ATP binding and/or hydrolysis. This is shown in the model as an arrowfrom NBD2 to NBD1. Once ATP has bound to both NBDs, ATP hydrolysis atNBD1 opens the channel to the bursting state. ATP bound to NBD2stabilizes the bursting state by allosterically stabilizing the openstate conformation at NBD1. Eventually, hydrolysis of ATP at NBD2 occurswhich destabilizes the bursting state, and the channel closes from thebursting state to the long closed state.

The fact that PP_(i) has been shown to bind to NBD of CFTR provides astrong precedent for PP_(i) binding to the NBDs of other ABC proteins,where it can mimic some aspects of ATP binding. PP_(i) most likelyexerts its effects by binding to NBD2. Previous study of CFTR mutantshas shown that NBD2 appears to regulate the duration of burst. Inaddition, NBD2 mutations altered the rate at which the channel opens.Both of these processes are influenced by PP_(i). Moreover, it has beenshown that addition of non-hydrolyzable ATP analogs can prolong theduration of bursts, an effect attributed to NBD2, suggesting that therate of hydrolysis regulates the duration of bursts.

In the model appearing in FIG. 2B, it is shown that PP_(i) cansubstitute for ATP in binding to NBD2. When PP_(i) binds, it mimics theeffect of ATP binding, allosterically increasing the binding (andperhaps hydrolysis) of ATP at NBD1 (indicated by the "+" in FIG. 2B).Hydrolysis of ATP at NBD1 then opens channel to the bursting state.While PP_(i) remains bound to NBD2, the bursting state is stabilized, asif ATP were present (third panel from top in FIG. 2B). Unlike ATP,however, PP_(i) is not hydrolyzed and the channel remains stabilized inthe bursting state for a prolonged period (indicated by "-" in FIG. 2B).In this case, termination of the burst may occur only when PP_(i) hasdissociated from NBD2.

Why PP_(i) would show preferential binding to NBD2 is not known, but thetwo NBDs do have significantly different amino acid sequences, and thereis substantial precedent for nucleotide-binding sites that showpreference between different analogs and PP_(i). The finding that 5 mMPP_(i) plus 0.3 mM ATP (Example 1) opens the channel more rapidly than0.3 mM ATP alone and the observation that PP_(i) stimulates binding of30 mM 8-N₃ ATP may be due to the high concentration of PP_(i) relativeto ATP. Perhaps the binding affinity or steric constraints of NBD2 aremore favorable for PP_(i) binding.

Thus, it is possible that the PP_(i) interacts primarily with NBD2 andin so doing it alters two functions of NBD2: a) it has an allostericeffect on NBD1, enhancing binding and/or hydrolysis of ATP and b)because it is not hydrolyzed it stabilizes the bursting state preventingchannel closure.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for treating or preventing a disease orcondition associated with inadequate ABC protein activity in a subjectcomprising administering to the subject an effective amount of aninorganic pyrophosphate (PPi).
 2. A method of claim 1, wherein thedisease is Cystic Fibrosis and the effective amount is an amountsufficient to activate mutant cystic fibrosis transmembrane conductanceregulator (CFTR) proteins in the subject.
 3. A method of claim 1,wherein the condition is constipation and the effective amount is anamount sufficient to activate wild-type cystic fibrosis transmembraneconductance regulator (CFTR) proteins in the subject.
 4. A method ofclaim 1, wherein the disease is selected from the group consisting ofinfantile Refsum disease, hyperpipecolic acidaemia, neonataladrenoleukodystrophy and Zellweger's Syndrome and the effective amountis an amount sufficient to activate PMP70 in the subject.
 5. A method ofclaim 1, wherein the disease is an autoimmune disease, which is based onMHC-linked TAP transport protein mediated antigen presentation, and theeffective amount is an amount sufficient to activate MHC-linked (TAP)transport proteins in the subject and the inorganic pyrophosphate isnon-hydrolyzable.
 6. A method of claim 1, wherein the condition is organtransplant rejection, and the effective amount is an amount sufficientto activate MHC-linked (TAP) transport proteins in the subject and theinorganic pyrophosphate in non-hydrolyzable.
 7. A method of claim 1,wherein the condition is chemotherapy resistance, and the effectiveamount is an amount sufficient to activate P-glycoprotein in the subjectand the inorganic pyrophosphate is non-hydrolyzable.